The purpose of this project is to isolate and characterize the cholesterol moiety that we have shown to be released by activated human and rat platelets. Platelet-free supernatants were prepared from incubated thrombin-activated human and rat platelets. Unique appearing cholesterol-phospholipid vesicles (greater than 600 angstroms in diameter) with numerous rod-shaped projections were isolated from these supernatants. Chemical analysis showed that vesicles released from activated human or rat platelets contained 9% cholesterol, 34% phospholipids, 1% triglycerides, and 56% protein. The molar ratio of cholesterol to phospholipid in these vesicles was 0.5, whereas, the molar ratio of cholesterol to phospholipid was 1.0 vesicles released from activated platelets obtained from rats fed a high-cholesterol diet. In addition, prolonged incubation of activated platelets (18 hrs rather than the usual 2 hrs) also resulted in vesicles with a substantially higher cholesterol to phospholipid molar ratio (0.9). All vesicles demonstrated a density of 1.14 when centrifuged isopycnically in a continuous sucrose density gradient. This research has been directed towards identifying how platelets can contribute directly or indirectly to the formation of atherosclerotic lesions which characteristically contain cells with large amounts of accumulated cholesterol. When macrophages were incubated with the platelet-derived cholesterol-containing vesicles, a substantial increase in their cholesteryl ester content was found in comparison with control cultures. Thus, these studies demonstrate that activated platelets release cholesterol that can be accumulated by cells. This process may represent a new mechanism to explain the deposition of cholesterol within cells of thrombi and possibly atherosclerotic lesions.